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-Dependent Neutrophil Reactive Oxygen Regulation of Adhesion-Independent andKinases Play a Nonredundant Role in Class I Phosphoinositide-3-Kinases and Src

A. Lowell, Emilio Hirsch and Giorgio BertonLaura Fumagalli, Carlo C. Campa, Giulia Germena, Clifford

http://www.jimmunol.org/content/190/7/3648doi: 10.4049/jimmunol.1201951February 2013;

2013; 190:3648-3660; Prepublished online 27J Immunol 

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The Journal of Immunology

Class I Phosphoinositide-3-Kinases and Src Kinases Playa Nonredundant Role in Regulation of Adhesion-Independentand -Dependent Neutrophil Reactive Oxygen SpeciesGeneration

Laura Fumagalli,* Carlo C. Campa,† Giulia Germena,† Clifford A. Lowell,‡

Emilio Hirsch,† and Giorgio Berton*

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very

rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases

(PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine

neutrophils deficient of PI3Kg or Hck, Fgr, and Lyn. Our studies show that PI3Kg is indispensable for the early, fMLF-induced

ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response

to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kg-deficient neutrophils. In

contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition

of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the

late one. Selective inhibitors of PI3Ka and PI3Kd markedly reduced both the early and late response to fMLF and TNF in human

neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kg via Ras in the early phase of the response and

by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in

suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for

Vav phosphorylation. The Journal of Immunology, 2013, 190: 3648–3660.

Myeloid leukocytes generate reactive oxygen species(ROS) upon interaction of a wide variety of ligandswith specific surface receptors due to the activation of

a multimeric NADPH oxidase, also referred to as NOX2, thattransfers electrons from NADPH to molecular oxygen (1). Besidesexerting microbicidal actions, ROS are cytotoxic and, via oxida-tive inactivation of tyrosine and serine/threonine phosphatases,

enhance protein kinase activities, leading to activation of tran-scription factors and amplification of inflammation (2).Early studies by Nathan and Ding (3) made the paradigm that

neutrophil ROS generation, in response to chemoattractants, oc-curs in two phases. The first phase is very rapid, transient, inde-pendent of cell adhesion to cell and extracellular-matrix integrincounterreceptors and enhanced by priming agents such as LPS,cytokines, and the F-actin depolimerizing mycotoxin cytochalasinB (CB). In contrast, the second phase ensues after several minutes,lasts up to 30–40 min, is strictly dependent on engagement ofneutrophil integrins (4), and inhibited by CB (reviewed in Ref. 5).A few cytokines, such as TNF, are able to trigger the late integrin-dependent response selectively.Studies in mice with the genetic deficiencies of various signal

transduction components have established the paradigm that theearly phase of neutrophil ROS generation in response to chemo-attractants is absolutely dependent on the g isoform of phos-phoinositide 3-kinase (PI3Kg) (6), whereas the adhesion, integrin-dependent late response requires Src-family kinases (SFKs) (7)and Syk (8), a cytoplasmic tyrosine kinase acting downstream ofSFKs (9, 10). Later studies suggested a more complex scenario,providing evidence that in primed neutrophils, also the PI3K IAisoforms a, b, and d may be stimulated by the chemoattractantfMLF, and SFKs may be implicated in this pathway (11, 12).Additionally, adhesion-dependent neutrophil ROS generation inresponse to TNF was reported to depend on class IA PI3Ks (13),and SFKs were implicated also in the early, adhesion-independentphase of fMLF-induced superoxide generation (14).Mechanisms by which PI3Ks and SFKs regulate NADPH oxi-

dase activity are only partly understood. Phosphatidylinositol phos-phorylated at the 3 position of the inositol ring (PIP3), formed byPI3Ks, provides a binding site for the PX domain of cytosolic

*Section of General Pathology, Department of Pathology and Diagnostics, Universityof Verona, Verona 37134, Italy; †Department of Genetics, Biology and Biochemistry,Molecular Biotechnology Center, University of Torino, Torino 10126, Italy; and‡Department of Laboratory Medicine, University of California, San Francisco, SanFrancisco, CA 94122

Received for publication July 13, 2012. Accepted for publication January 31, 2013.

This work was supported by grants from Ministero dell’Istruzione, dell’Universita edella Ricerca of Italy (2009 Progetti di ricerca di rilevante interesse nazionale grant toG.B. and E.H.) and Fondation Leducq 09CVD01 (to E.H.). The research leading toresults concerning the role played by Src kinases in regulating neutrophil responsesreceived funding from the European Community’s Seventh Framework Programme(FP7-2007-2013) under Grant Agreement HEALTH-F4-2011-282095-TARKINAID(to G.B.).

Address correspondence and reprint requests to Prof. Giorgio Berton and Dr. EmilioHirsch, Section of General Pathology, Department of Pathology and Diagnostics,Strada Le Grazie 8, Universita degli Studi di Verona, Verona 37134, Italy (G.B.)and Department of Genetics, Biology and Biochemistry, Molecular BiotechnologyCenter, Via Nizza 52, Torino 10126, Italy (E.H.) E-mail addresses: giorgio.berton@univr.it (G.B.) and emilio.hirsch@unito.it (E.H.)

The online version of this article contains supplemental material.

Abbreviations used in this article: CB, cytochalasin B; GEF, guanine nucleotideexchange factor; HGCa, HBSS supplemented with 0.5 mM CaCl2 and 5 mM D-glucose; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol phosphorylatedat the 3 position of the inositol ring; PLC, phospholipase C; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; ROS, reactive oxygen species;SFK, Src-family kinase.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

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NADPH oxidase components (1). Activation of the Ser/Thr kinaseAKT following its binding to PIP3 via its pleckstrin homologydomain results in phosphorylation of p47phox in its autoinhibitoryregion (1, 15). Finally, PIP3 formation by PI3Ks regulates ac-tivation of at least three different guanine nucleotide exchangefactors (GEFs) for the small GTP-binding protein Rac, a compo-nent essential for activation of NADPH oxidase (1, 16–18). No-tably, neutrophils deficient of one of these three GEFs (i.e., P-Rex1,Vav proteins, and DOCK2) are markedly defective in NADPHoxidase activation (19–23).Mechanisms of activation of NADPH oxidase by SFKs are

poorly understood. In the context of the classical chemoattractant-induced early phase of ROS generation, SFKs have been reportedto regulate activation of PI3Ks (11, 12, 24). Additionally, SFKsare required for chemoattractant-induced Vav phosphorylation andRac activation and neutrophils deficient of Vav1 or Hck and Fgrhave a defect in NADPH oxidase activation by fMLF (14, 19).Class IA PI3Ks and Vav proteins have been reported to also reg-ulate the integrin-dependent phase of neutrophil activation (12,13, 25–27). An additional important substrate of the integrin/SFKs/Syk signaling pathway regulating this late phase of ROSgeneration has been recently identified as phospholipase C (PLC)g2 (28).In this report, we addressed the role of PI3Ks and SFKs in the

early and late phase of ROS generation by unprimed human andmurine neutrophils in response to the chemoattractant fMLF andTNF. Using inhibitory compounds, we found that PI3Kg and SFKsplay essential and nonredundant roles in activation of the early andthe late phases of ROS generation in human neutrophils. Defi-ciency of PI3Kg resulted in a total suppression of the early phaseof ROS generation in response to fMLF in mouse neutrophils butdid not affect the adhesion-dependent late phase in response todifferent agonists. Conversely, the combined deficiency of Hck,Fgr, and Lyn resulted in an almost total or total suppression ofboth the early and the late phases of ROS generation, respectively,an effect that, based on the use of selective inhibitors, was largelydependent on the class IA PI3Ks PI3Ka and PI3Kd, as well as theRac GEF Vav. Collectively, these findings help define the mech-anisms of regulation of ROS production by PI3Ks and SFKs.

Materials and MethodsCell preparation

Generation and maintenance of hck2/2fgr2/2lyn2/2 triple-knockout miceand PI3Kg knockout mice in the C57BL/6J background were as described(6, 29). Wild-type and knockout animals used in the experiments were at8–10 wk of age. Animals were housed at a pathogen-free facility at theUniversity of Verona or Turin and treated according to protocols approvedby the Minister of Health of Italy and the university animal care com-mittee. Mouse bone marrow neutrophils were isolated by centrifugation ofbone marrow cells flushed from femurs and tibias over a Percoll discon-tinuous density gradient (Amersham, Arlington Heights, IL) exactly asdescribed previously (14). After isolation, neutrophils were washed in Ca2+/Mg2+-free HBSS then resuspended at 10 3 106/ml in HBSS supplementedwith 0.5 mM CaCl2 and 5 mM D-glucose (HGCa). Routinely, cell sus-pensions were left at room temperature for 1 h before assay. Human PMNswere prepared from buffy coats of healthy volunteers by centrifugationthrough Ficoll Paque Plus (Amersham). Patients provided their informedconsent before samples were taken. Contaminating erythrocytes were re-moved by Dextran 500 (Amersham) sedimentation followed by hypotoniclysis. After isolation, cells were suspended in HGCa at 10 3 106/mland left at room temperature for 1 h before assay. Neutrophils wereeither left untreated or incubated for 10 min at 37˚C with 10 mM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)(Calbiochem, Darmstadt, Germany), 100 nM wortmannin (Calbiochem),1 mM diphenyleneiodonium chloride (Calbiochem), or 50 U/ml superoxidedismutase (Sigma-Aldrich, St. Louis, MO). For experiments with selectiveinhibitors of different PI3K isoforms, human neutrophils were pretreatedfor 10 min at room temperature with the PI3Kg inhibitor AS604850

(Alexis Biochemical, San Diego, CA), the PI3Ka inhibitor compound 15e(Alexis Biochemical), the PI3Kb inhibitor TGX22 (Alexis Biochemical),the PI3Kd inhibitor IC87114 (Symansis, Auckland, New Zealand), or thebroad-specificity PI3K inhibitor compound PI103 (Cayman Chemical,Montigny-le-Bretonneux, France) at the concentration indicated in the text.

Measurement of ROS production

Isoluminol-based chemiluminescence assays (30) were performed in HGCain the presence of HRP and isoluminol (both from Sigma-Aldrich) in atemperature-controlled Multilabel Count Victor X5 (PerkinElmer). Assayswere run in dark 96-well plates precoated with 250 mg/ml human fibrin-ogen (14) in HGCa containing (final concentration): 100 mM isoluminoland 8 U/ml HRP. After addition of 13 105 human neutrophils or 7.53 105

mouse neutrophils, plates were left at 37˚C for 5–10 min and the reactionstarted by addition of the stimulus (see text for concentrations). Lightemission was recorded every minute.

Active Ras pulldown assay

Measurement of active RAS (Ras-guanosine triphosphate) was carried outusing a RAS Activation assay kit (EMD Millipore; Merck, Darmstadt,Germany) according to the manufacturer’s instructions. Neutrophils werestimulated in suspension with 5 mM fMLF, and, at different time points,cells were lysed in ice cold lysis buffer. Cell lysates were centrifuged at4˚C for 5 min at 13,000 rpm, and the supernatant was incubated withglutathione GST-RAF-RBD coupled to glutathione agarose for 1 h at 4˚C.Proteins bound to the beads were washed three times in lysis buffer, andthe amount of bound proteins was quantified by Western blot analysis.

Western blotting

Murine or human neutrophils (10 3 106/ml) in HGCa were plated in 24-well plates precoated with human fibrinogen (Sigma-Aldrich) and stimu-lated with fMLF (see text) or human (10 ng/ml) (PeproTech, Rocky Hill,NJ) or murine TNF (5 ng/ml) (BioSource International, Camarillo, CA) forthe time indicated in the Results. At the appropriate time, cell activationwas stopped by addition of a half volume of ice-cold HBSS (without Ca2+

and glucose) containing a 3-fold concentration of protease inhibitors(Roche Molecular Biochemicals, Mannheim, Germany) supplementedwith 3 mM Na3VO4, 30 mM phenylarsine oxide (Sigma-Aldrich), and3 mM diisopropyl-fluorophosphate (Sigma-Aldrich). Samples were kept inice for 10 min before lysis with 43 Sample Buffer. Samples were separatedon SDS-PAGE gels and transferred to nitrocellulose Hybond C (Amersham).After quenching with 3% BSA in TBS for 1 h, blots were incubatedovernight at 4˚C with primary Abs, followed by HRP-conjugated donkeyanti-rabbit or goat anti-mouse Abs (Amersham). Immunoreactivity wasdetected using Immobilon Western detection reagent (Millipore) and ana-lyzed with a Quant Image LAS400 (GE Healthcare). Abs used in this studywere as follows: anti-phosphospecific Ab directed against Akt (Ser473),p38 MAPK (Thr180/Tyr182), and ERK 1/2 MAPK (Thr202/Tyr204) werefrom Cell Signaling Technology (Beverly, MA). Anti-protein Abs directedagainst Akt and p38 were from Cell Signaling Technology. Anti-proteinAbs directed against ERK1/2 were from Santa Cruz Biotechnology. Anti-Vav Abs were from Upstate Biotechnology, and anti-phosphospecific Abdirected against Vav (Y174) was from EnoGene Biotech (New York, NY).

Cell spreading

Human or murine neutrophils in HGCawere plated in 24-well tissue-cultureplastic in the absence of any stimulus. Photos were taken with a340 phase-contrast objective with a Digital Compact Olympus (Olympus, Hamburg,Germany) camera.

Statistics

The Student t test has been applied to examine the statistical significance ofdifferences between most of the data with the exception of the Ras activationdata, which were examined by the two-way ANOVA with Bonferroni posthoc test. Values of *p , 0.05 or **p , 0.01 were taken as significant.

ResultsROS generation in response to fMLF by unprimed humanneutrophils occurs in two distinct phases that require PI3K andSFK activities

In this study, ROS generation was assayed with a very sensitivechemiluminescence assay (30) that allowed us to monitor neu-trophil ROS generation in the absence of any priming agent.

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fMLF-stimulated ROS generation by human unprimed neutrophilsplated in fibrinogen-coated well plates occurred in two phases(Supplemental Fig. 1A–D). One was very rapid and, after a peak at∼0.5–1 min, declined rapidly. The second phase became detect-able within 5–10 min and steadily increased up to 25–30 minwhen a plateau was reached. The detected chemiluminescencesignal was due mainly to the extracellular generation of super-oxide anion by the NADPH oxidase because addition of super-oxide dismutase, which catalizes the dismutation of superoxide tohydrogen peroxide, significantly suppressed the response (Sup-plemental Fig. 1A). Addition of diphenyleneiodonium chloride, anNADPH oxidase inhibitor, completely blocked the response.As predicted from previous studies (see Ref. 14 and references

contained therein), inhibition of SFKs by PP2 (Supplemental Fig.1B) or PI3Ks using broad specificity inhibitors such as wort-mannin (Supplemental Fig. 1C) or PI-103 (Supplemental Fig. 1D)totally inhibited both the early and the late adhesion-dependentresponse of human neutrophils to fMLF. Notably, the dependenceof these responses on both SFKs and PI3Ks seems to be a generalfeature of signal transduction pathways ensuing from chemotacticpeptide receptors, because similar results were obtained using C5ainstead of fMLF (Supplemental Fig. 1E). Consistent with previousfindings, PMA-induced ROS generation was not inhibited by PP2or wortmannin (Supplemental Fig. 1F).

SFKs are not implicated in activation of class IA PI3Ks in thecourse of the early response to fMLF

Recent studies have demonstrated that class IA PI3Ks may be ac-tivated, either directly or indirectly, by trimeric G protein–coupledreceptors (see Ref. 31). Notably, in TNF-primed neutrophils, fMLFtriggers two phases of PIP3 formation, the second of which peaksat 1 min, is dependent on class IA PI3Ks, and inhibited by the SFKinhibitor PP1 (11). We therefore examined the effect of a SFKinhibitory drug or SFK genetic deficiency on phosphorylation ofthe PI3K downstream target AKT in human or murine neutro-phils, respectively (Fig. 1). In our assay conditions, fMLF trig-

gered a robust AKT phosphorylation on the S473 residue within1 min, which then declined but remained detectable up to 30 minin human neutrophils. Whereas in wortmannin-treated neutrophilsAKT phosphorylation was totally blunted at all time points tested(Fig. 1A), the SFK inhibitor PP2 only slightly decreased AKTphosphorylation at 1 min, but had a strong inhibitory effect at latertime points (Fig. 1B). In three distinct experiments (Fig. 1C), AKTphosphorylation in PP2-treated neutrophils was 69.0 6 6.9% at1 min, compared with untreated cells; however, this difference wasnot statistically significant. Consistently, deficiency of Hck, Fgr,and Lyn, the three SFKs expressed at the highest levels in neu-trophils, had a minor effect on fMLF-induced AKT phosphory-lation at 1 min (Fig. 1D), but resulted in a significantly reducedAKT phosphorylation at later time points. We conclude that ac-tivation of PI3Ks occurring in the first phase of oxidant generation(within 1 min) is mainly independent of SFK activity. However,PI3K activity preceding, or concomitant with, the second adhesion-dependent phase of oxidant generation requires SFKs.

PI3Kg regulates activation of Ras and phosphorylation of theRas downstream target ERK proteins

Because class IA PI3Ks may also be activated via an increase inthe GTP-bound form of Ras (31), and fMLF has been demon-strated to activate GDP/GTP exchange on Ras (32), we examinedfMLF-stimulated phosphorylation of the Ras downstream targetERK proteins and the dependence of these response on PI3K andSFK activities in human and murine neutrophils. fMLF triggereda rapid, but transient, phosphorylation of ERK proteins that peakedat 1 min and then declined to background levels in human neu-trophils (Fig. 2). Consistent with previous reports (12, 14), ERKphosphorylation was not affected by PP2 (Fig. 2A), but markedlyinhibited by wortmannin (Fig. 2B). fMLF also induced tyrosinephosphorylation of another MAP kinase member, p38, whichpeaked, similar to ERK phosphorylation, at 1 min of stimulation;however, neither PP2 nor wortmannin had any effect on this earlyp38 phosphorylation (Supplemental Fig. 2A, 2B).

FIGURE 1. Different roles of PI3K and SFK activities in fMLF-induced AKT phosphorylation. Human (A–C) or murine (D) neutrophils were plated in

fibrinogen-coated wells and stimulated with 1 or 5 mM fMLF for human or mouse cells, respectively, in the presence or the absence of PP2 or wortmannin

as described in Materials and Methods. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the

indicated specificity as described in Materials and Methods. (C) reports densitometric analysis, expressed as ratio between the phosphoprotein versus the

total specific protein signal, of AKT phosphorylation at 1 min after the addition of fMLF in neutrophils treated or not with 10 mM PP2. Mean results 6 SD

of three independent experiments are reported. In (E), histograms at the right of the immunoblot report densitometric analysis, expressed as ratio between

the phosphoprotein versus the total specific protein signal, of AKT phosphorylation in wild-type versus hck2/2fgr2/2lyn2/2 neutrophils. Mean results 6SD of three independent experiments are reported. *p , 0.05.

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The time course of fMLF-stimulated ERK phosphorylation inmurine neutrophils differed from that detected in human cells,displaying a maximum at 5 min (Fig. 2). ERK phosphorylation at1 min was defective in PI3Kg2/2 neutrophils (Fig. 2C), but not incells deficient of Fgr, Hck, and Lyn (Fig. 2E). Notably, at this timepoint, AKT phosphorylation was not affected by SFK deficiency(see Fig. 1D), but totally defective in PI3Kg2/2 neutrophils (Fig.2D). At the 5-min time point after fMLF stimulation, both SFK andPI3Kg deficiency resulted in a marked decrease of ERK phos-phorylation, suggesting that they may play nonredundant roles inactivation of the Ras pathway. Notably, we reproducibly found thatPI3Kg deficiency causes a marked decrease in AKT phosphoryla-tion also at the later time points of 30 min (Fig. 2D). Although, wedid not address this issue in detail, this finding suggests that, in-dependently of the mechanism involved, prolonged AKT phos-phorylation in response to fMLF requires PI3Kg (see Discussion).We also examined the effect of a selective inhibitor of PI3Kg,

the AS604850 compound, on ERK phosphorylation in human neu-trophils. At 1 mM, AS604850 inhibited the early PI3Kg-dependentphase of ROS generation, but not the late, adhesion-dependent, andPI3Kg-independent one (see below). Notably, at this same con-centration, AS605240 markedly inhibited (1-min time point) ortotally suppressed (5-min time point) both AKT and ERK phos-phorylation (Fig. 2F, 2G).A very recent report demonstrated that fMLF activates PI3Kg

also downstream a PLCb/diacylglycerol pathway that impinges onthe Ras GEF RasGRP4, thus activating Ras (33). To strengthen theevidence emerging from our studies (Fig. 2) that positive feedbackcircuits of PI3K activation may also include a PI3Kg/Ras, besidesa Ras/PI3Kg, pathway (33), we addressed whether fMLF-inducedRas activation is defective in PI3Kg2/2 neutrophils (Fig. 3). Wefound that deficiency of PI3Kg resulted in a marked inhibition ofGTP loading on Ras at 30 s after fMLF stimulation (i.e., at a timepoint preceding the peak of the early wave of ROS generation).Taken together, the data reported in Figs. 1–3 suggest that PI3Kg

plays a major role in the early response to fMLF and, via activationof Ras, it may establish a positive-feedback loop of PI3K activationimplicating the class IA isoforms. In contrast, SFKs play onlya marginal role in the regulation of PI3K activity in response tofMLF that occurs within 1 min following stimulation, which iscoincident with the first wave of ROS generation (see Fig. 4 andSupplemental Fig. 1).

The g isoform of class I PI3Ks is indispensable for the earlyphase of ROS generation in response to fMLF, but plays no rolein the late, adhesion-, and SFK-dependent one

To better elucidate the role of class IA and class IB PI3Ks andSFKs in signal transduction pathways implicated in the two wavesof ROS generation in response to fMLF, we examined this responsein neutrophils with the genetic deficiency of PI3Kg and SFKs. Asreported in Fig. 4A, and in accord with previous studies in CB–primed hck2/2fgr2/2 neutrophils (14), the early, fMLF-inducedwave of ROS generation was markedly reduced in triple-knockouthck2/2fgr2/2lyn2/2 neutrophils and, as shown in human cells(Supplemental Fig. 1B), totally suppressed by PP2 (data not shown).Notably, the integrin-dependent wave of oxidant generation dis-played a more stringent dependency on SFK activities, and, accord-ing to previous data with hck2/2fgr2/2 neutrophils (7), it was totallysuppressed in hck2/2fgr2/2lyn2/2 neutrophils (Fig. 4A).Examining PI3Kg-deficient neutrophils, we found that, as ex-

pected from previous studies (6), there was a total lack of theearly response to fMLF (Fig. 4B). In contrast, the late phase ofROS generation in wild-type and PI3Kg2/2 neutrophils wascomparable. Consistently, treatment of human neutrophils with the

selective PI3Kg inhibitor AS604850 at 1 mM concentration sig-nificantly inhibited the early, but not the late, adhesion-dependentphase of ROS generation (Fig. 4C).

Adhesion-dependent, TNF-stimulated ROS generation byneutrophils requires SFKs but is independent of PI3Kg

To strengthen the above-described findings suggesting that SFKs,but not PI3Kg, are indispensable for the late adhesion-dependentphase of oxidant generation, we examined the response to TNF, anagonist that triggers only the late, adhesion-dependent phase ofneutrophil activation (3, 5, 7).Experiments with human neutrophils treated with SFK or PI3K

inhibitors showed a total dependence of the response to TNF onthese kinases (Supplemental Fig. 3A, 3B). Despite some variabilityat the early time points (compare Supplemental Fig. 3C–E), thetime course of this response was coincident with the phosphory-lation of the PI3K downstream target AKT, being detectable atleast after 5 min of stimulation (Supplemental Fig. 3C), a findingconfirming previous studies (13), but undetectable at earlier timepoints (see Supplemental Fig. 3C–E). This delayed TNF-inducedAKT phosphorylation was almost totally suppressed by PP2 andwortmannin in human neutrophils (Supplemental Fig. 3D, 3E). Inmurine neutrophils, TNF-induced AKT phosphorylation was lessstrong than that induced by fMLF (Supplemental Fig. 3F, 3E,compare with Figs. 1D, 2D, respectively). However, whereas de-ficiency of PI3Kg did not substantially reduce AKT phosphory-lation at 5 and 30 min from stimulation, deficiency of Hck, Fgr,and Lyn reduced AKT phosphorylation at these time points(Supplemental Fig. 3G).We then examined the response of murine neutrophils plated on

fibrinogen and stimulated with TNF or plated on plain tissue-culture plastic in the absence of any stimulus (Fig. 5). In theseassay conditions, ROS generation by SFK-deficient neutrophils istotally defective (7 and data not shown). In contrast, deficiency ofPI3Kg did not significantly affect ROS generation by neutrophilsstimulated with TNF (Fig. 5A) or plated on plain plastic in theabsence of any stimulus (Fig. 5B).Because ROS generation by neutrophils plated on fibrinogen in

the presence of TNF or on plain plastic is accompanied by cellspreading over the adherence surface, a response that was reportedto depend on b2 integrins (34), we also examined neutrophilspreading on plastic. Wild-type murine neutrophils spread nicelyon plastic (Fig. 5C), and deficiency of Hck, Fgr, and Lyn resultedin a total absence of this response (Fig. 5D). Notably, spreading ofPI3Kg-deficient neutrophils on plastic was comparable to that ofwild-type cells (Fig. 5E), but totally suppressed by wortmannin(Fig. 5F). The selective PI3Kd inhibitor IC87114 reduced neu-trophils spreading in wild-type (Fig. 5G) and PI3Kg-deficient(Fig. 5H) neutrophils, suggesting that this class IA PI3K isoform,together with other wortmannin-sensitive PI3Ks, regulates neu-trophil spreading. A quantitation of surface area of murine neu-trophils plated on tissue-culture plastic confirmed that selectivePI3Ka and d inhibitors reduced spreading of both wild-type andPI3Kg2/2 murine neutrophils (Table I). In contrast, a PI3Kb in-hibitor did not inhibit or only marginally inhibited spreading ofwild-type or mutant neutrophils, respectively.The dependence on class IA, but not class IB, PI3Ks of integrin-

dependent spreading was confirmed by studies with human neu-trophils treated with selective PI3K inhibitors (Supplemental Fig. 4).In fact, spreading of human neutrophils on plastic in the absence ofany stimulus or on fibrinogen in the presence of TNF was markedlyinhibited by PI3Ka and d inhibitors. In contrast, a PI3Kg inhibitordid not affect neutrophil spreading on plastic or only marginallyaffected it on fibrinogen in the presence of TNF.

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FIGURE 2. Different roles of PI3K and SFK activities in fMLF-induced ERK phosphorylation. Human or murine neutrophils were stimulated in the presence or

the absence of the indicated inhibitors and lysed as described in Fig. 1 andMaterials and Methods. Lysates were subjected to immunoblot analysis with Abs of the

indicated specificity as described in Materials and Methods. Human neutrophils stimulated with 1 mM fMLF in the presence or the absence of PP2 (A) or

wortmannin (B). Graphs to the right of the immunoblots report densitometric analysis, expressed as ratio between the phosphoprotein versus the total ERK protein

signal. One representative experiment of three to four performed with identical results is reported. (C and D) Wild-type or PI3Kg2/2 murine neutrophils were left

untreated or stimulated with 5 mM fMLF and lysed at the time points indicated. Graphs to the right of the immunoblots report densitometric analysis expressed as

ratio between the phosphoprotein versus the total ERK (C) or AKT (D) protein signal. Mean results6 SD of three independent experiments are reported. (E) Wild-

type or hck2/2fgr2/2lyn2/2 neutrophils were stimulated as described above for analysis of ERK phosphorylation. Graphs to the right of the immunoblots report

densitometric analysis expressed as ratio between the phosphoprotein versus the total ERK protein signal. Mean results6 SD of three independent experiments are

reported. (F and G) Human neutrophils were stimulated with 1 mM fMLF in the presence or the absence of 1 mM AS604850 for analysis of AKT and ERK

phosphorylation. One representative experiment of two to three performed with identical results is reported. *p , 0.05, **p , 0.01.

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PI3Ka and d are implicated in adhesion-dependent ROSgeneration

To identify PI3Ks responsible for the late, adhesion-dependentphase of ROS generation, we used inhibitors that have been re-ported to display some selectivity for different class IA PI3Ks (35).At 40 nM concentration, the compound TGX-221, which shouldselectively inhibit the PI3Kb isoform by ∼85% (26), had someinhibitory effect on the response to TNF (Fig. 6A) and both theacute and the late response to fMLF (Fig. 6B). However, data oftotal light units integrated over 2 min for the acute and 5–30 minfor the late adhesion-dependent response showed that differencesbetween untreated and drug-treated neutrophils were not statisti-cally significant (Fig. 6C). This finding agrees with the evidencethat TGX-221 only marginally inhibited murine (Table I) or hu-man (data not shown) neutrophil spreading. In contrast, compound15e, a relative selective inhibitor of PI3Ka, and compoundIC87114, a relatively selective inhibitor of PI3Kd, used at the lowdoses of 0.1 and 0.5 mM, respectively, markedly suppressed theresponse to TNF (Fig. 6D, 6G). Data of total light units integratedover 5–30 min (Fig. 6F, 6I) showed that the response to TNF wasinhibited by 76 6 16% (n = 6) and 87 6 14% (n = 6) by thecompound 15e and IC87114, respectively.Examining the effects of these two inhibitors on the response to

fMLF (Fig. 6E, 6H), we found that both the early and the lateresponse were significantly inhibited. Using different doses ofcompound 15e and IC87114 on fMLF-induced ROS generationdid not allow us to find a concentration at which only the lateadhesion-dependent response was inhibited (data not shown). Weconclude that, also in our experimental conditions, PI3Ka and/orPI3Kd are implicated in the early response to fMLF.

SFKs are essential for the early and late phase of Vavphosphorylation in response to fMLF and TNF

Results described so far suggest that PI3Ks and SFKs play a co-ordinated but nonredundant role in regulation of neutrophil ROSgeneration. Because GTP loading on Rac is indispensable foractivation of the NADPH oxidase and, among the three Rac GEFsimplicated in Rac activation in neutrophils (see Introduction), onlyVav is known to be directly activated downstream SFKs (36), weaddressed the effects of SFK inhibitors and SFK deficiency on Vavphosphorylation in human and murine neutrophils, respectively(Fig. 7). Specifically, we examined phosphorylation of Y174, theVav1 residue that in its unphosphorylated form dictates intra-molecular inhibitory interaction between the Vav acidic (Ac) motifand the DH domain; phosphorylation of Y174, or analogous res-idues of Vav 2/3, releases this interaction and allows access of theDH domain to its target (36). As shown in Fig. 7A, fMLF trig-gered a rapid Y174 phosphorylation of Vav that increased up to 30min [i.e., within the time frame of ROS generation (see Supple-mental Fig. 1) in human neutrophils]. In contrast, TNF-inducedVav phosphorylation was delayed (Fig. 7B), but, similar to thefMLF-induced response, coincident with the time course of ROS

generation (see Supplemental Fig. 3). Notably, PP2 totally sup-pressed Vav tyrosine phosphorylation in response to fMLF or TNFin human neutrophils at all of the time points tested. Time coursesof Vav phosphorylation in response to fMLF or TNF in wild-typemurine neutrophils were similar to those of human cells, butneutrophils deficient of Hck, Fgr, and Lyn were totally defective inVav phosphorylation at all time points tested and independently ofthe stimulus used (Fig. 7C).Having established that SFKs are essential for Vav phos-

phorylation in both the early adhesion-independent and the lateadhesion-dependent phase of neutrophil responses, we addressedthe role of PI3Ks in the regulation of Vav phosphorylation usingbroad specificity and selective PI3K inhibitors. As shown in Fig.7D, wortmannin suppressed fMLF-induced Vav phosphorylationin the course of both the early and late response; in contrast,the late TNF-induced response was unaffected by wortmannin.Studies with selective class IA inhibitors showed that both PI3Kaand PI3Kd regulate the late adhesion-dependent phase of Vavphosphorylation in response to fMLF. In fact, the PI3Kd inhibitorIC87114 alone partially reduced Vav phosphorylation at the 15-and 30-min time points after fMLF stimulation (Fig. 7E), and thislate fMLF-induced response was more markedly reduced when itwas used in combination with the PI3Ka inhibitor compound 15e(Fig. 7F). However, consistent with the data obtained with wort-mannin, the PI3Kd inhibitor alone or in combination with thePI3Ka inhibitor did not affect the late response to TNF. Thesefindings suggest that in human neutrophils, Vav phosphorylation isregulated by PI3Ks in a stimulus-dependent manner, and TNF-induced Vav phosphorylation is largely independent of PI3K ac-tivities. The clear dissociation between inhibition of ROS gener-ation by class IA PI3K inhibitors (Fig. 6) and Vav phosphorylationsuggest that PIP3 formation is required for activation of additionalRac GEFs (see Ref. 23 and references contained therein).

DiscussionIn this report, we addressed signal transduction pathways regu-lating neutrophil ROS generation in an experimental setting mir-roring cell responses occurring in the context of neutrophiladhesion to the vascular endothelium and/or the interstitium.Agonists of trimeric G protein–coupled receptors trigger a veryrapid and transient generation of ROS. These same agonists, orsome cytokines, induce a slower, but prolonged ROS generation,which depends on adhesive interactions mediated by integrins (seeIntroduction and Ref. 5). This modality of ROS generation isbelieved to play an important role in the induction of tissuedamage in inflammatory pathology. Our findings show that dif-ferent PI3K isoforms and SFKs play an essential and not redun-dant role in regulating both phases of ROS generation (see Fig. 8for a model).As previously demonstrated (6), the class IB PI3K isoform

PI3Kg plays an essential role in stimulation of neutrophil ROSgeneration by fMLF in murine neutrophils. Neutrophils deficient

FIGURE 3. PI3Kg deficiency results in re-

duced Ras activation. Neutrophils were isolated

from wild-type or PI3g knockout mice and

Ras activation after fMLF (5 mM) stimulation

assayed as described in Materials and Methods.

(A) Representative detection of active Ras and

total Ras is shown. (B) Mean results of Ras

activation obtained in four independent experi-

ments. Values were normalized by setting the

percentage stimulation of PI3Kg+/+ cells at time

0 s to 1. *p , 0.05.

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FIGURE 4. Different roles of SFKs and PI3Kg in regulation of the early and late phase of fMLF-induced ROS generation in murine neutrophils. (A and

B) Neutrophils were isolated from wild-type, hck2/2fgr2/2lyn2/2 knockout or PI3g knockout mice as described in Materials and Methods and plated in

fibrinogen-coated wells. Cells were stimulated with 5 mM fMLF, and chemiluminescence was recorded every 1 min and up to 40 min. Mean results6 SD of

three to four independent experiments are reported. Graphs in the right panel report the total chemiluminescence detected during the early (0–2 min) or the

late (5–40 min) phase of ROS generation. (C) Human neutrophils were stimulated with 1 mM fMLF in the presence or the absence of 1 mM AS604850.

Mean results 6 SD of four independent experiments are reported. Graph in right panel reports the total chemiluminescence detected during the early (0–2

min) or the late (5–30 min) phase of ROS generation. *p , 0.05, **p , 0.01. KO, Knockout; WT, wild-type.

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of PI3Kg (Fig. 4) are virtually unable to generate ROS in re-sponse to fMLF. Together with previous evidence with TNF-primed neutrophils (11), this finding points to the conclusion thatPI3Kg play an indispensable role in the early phase of ROSgeneration triggered by chemoattractants. However, relatively se-lective inhibitors of PI3Ka and PI3Kd reduced the early fMLF-induced phase of ROS generation (Fig. 6), confirming previous

reports (11, 25, 37) on the implication of class IA PI3Ks in theresponse to chemoattractants.Also SFKs are indispensable for activating ROS generation by

fMLF. Early and more recent studies (14, 38) demonstrated a cleardefect in responses to fMLF by neutrophils deficient of Hck andFgr or Hck, Fgr, and Lyn. The early fMLF-induced ROS gener-ation is suppressed by the SFK inhibitor PP2 in human neutrophils

FIGURE 5. Late, TNF-induced

ROS generation is independent of

PI3Kg. Neutrophils were isolated

from wild-type or PI3g knockout

mice as described in Materials and

Methods and plated in fibrinogen-

coated wells in the presence of 5 ng/

ml TNF (A) or in tissue-culture plas-

tic wells in the absence of any stim-

ulus (B). Mean results 6 SD of four

independent experiments are reported.

(C–F) Neutrophils were isolated

from wild-type, hck2/2fgr2/2lyn2/2

knockout, or PI3g knockout mice as

described in Materials and Methods

and plated in tissue-culture plastic

wells in the absence of any stimulus.

Note that whereas both wild-type and

PI3g knockout neutrophils spread

on plastic, either deficiency of Hck,

Fgr, and Lyn or treatment with wort-

mannin totally suppressed spreading.

Wild-type (G) or PI3g knockout (H)

neutrophils were plated in tissue-cul-

ture plastic wells in the absence of

any stimulus and in the presence of

0.5 mM IC 87114. Photos were taken

with a 340 objective after 20 min.

KO, Knockout; WT, wild-type.

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(Supplemental Fig. 1) and markedly defective in Hck, Fgr, andLyn-deficient neutrophils (Fig. 4). Combined with previous find-ings (11), one possible explanation of these results is that SFKsregulate class IA PI3Ks, establishing a positive-feedback circuit ofPIP3 formation. However, examination of the PI3K downstreamtarget AKT led us to conclude that the role played by SFKs inregulation of this response is mainly independent of activation ofclass IA PI3Ks. Indeed, PP2 only slightly inhibited AKT phos-phorylation at an early time point after addition of the stimulus, andthis was not defective in hck2/2fgr2/2lyn2/2 neutrophils (Fig. 1).

An alternative mechanism of triggering of a positive-feedbackcircuit of PI3K activation is based on activation of Ras and itsdirect interaction with PI3K catalytic subunits (31). Examiningphosphorylation of a Ras downstream target, the ERK proteins,we found that this is almost totally suppressed by the broad-specificity PI3K inhibitor wortmannin and the PI3Kg selectiveinhibitor A604850 in human neutrophils and by the lack of PI3Kgexpression in murine neutrophils (Fig. 2). Together with the evi-dence that fMLF activates GDP/GTP exchange on Ras (32) andthis is defective in PI3Kg-deficient neutrophils (Fig. 3), these

FIGURE 6. Inhibitors of the a and d isoforms of class IA PI3Ks inhibit the early and late phase of ROS generation by human neutrophils. Human

neutrophils were plated in fibrinogen-coated wells and stimulated with 10 ng/ml TNF (A, D, G) or 1 mM fMLF (B, E, H) in the absence or the presence of 40

nM TGX-221 (A–C), 0.1 mM compound 15e (D–F), or 0.5 mM IC87114 (G–I). Chemiluminescence was recorded every 1 min and up to 40 min. Mean

results6 SD of five experiments are reported. (C, F, and I) Histograms report the total chemiluminescence detected during the early (0–2 min) or late (5–30

min) phase of ROS generation in the absence or the presence of the indicated inhibitor. *p , 0.05, **p , 0.01.

Table I. PI3Ka and d inhibitors reduce murine neutrophil spreading on uncoated tissue-culture plastic

Mouse Strain Untreated + IC87114 (PI3Kd Inhibitor) + Compound 15e (PI3Ka Inhibitor) + TGX-221 (PI3Kb Inhibitor)

Wild-typea 98.6 6 24.3 69.7 6 15.0*** 57.1 6 7.5*** 96.7 6 29.7 (NS)PI3Kg2/2a 114.5 6 22.0 86.9 6 25.0** 63.0 6 6.5*** 89.0 6 21.3*

Mean areas (6 SD) from $50 analyzed cells in two separate experiments are reported. Data are expressed in mm2.aMean area of both wild-type and PI3Kg2/2 neutrophils plated on plastic in the presence of wortmannin [i.e., in condition totally inhibiting cell

spreading (see Fig. 5)] was ∼50 mm2.*p , 0.05, **p , 0.01, ***p , 0.001.

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FIGURE 7. SFKs are indispensable for phosphorylation of Vav during the early and the late phase of ROS generation. Human neutrophils were plated in

fibrinogen-coated wells and stimulated with 1 mM fMLF (A) or 10 ng/ml TNF (B) in the presence or the absence of PP2 as described in Materials and

Methods. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the indicated specificity as de-

scribed in Materials and Methods. One representative experiment of three performed with identical results is reported. (C) Neutrophils were isolated from

wild-type or hck2/2fgr2/2lyn2/2 knockout mice as described in Materials and Methods and plated in fibrinogen-coated wells. Cells were stimulated with 5

mM fMLF or 5 ng/ml TNF. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the indicated

specificity. Graphs to the right of the immunoblot report densitometric analysis, expressed as ratio between the phosphoprotein versus the total Vav protein

signal. Mean results 6 SD of three independent experiments are reported. *p , 0.05, **p , 0.01, ***p , 0.001 (D–F). Human neutrophils were plated in

fibrinogen-coated wells and stimulated with 10 ng/ml TNF or 1 mM fMLF in the absence or the presence of 100 nM wortmannin (D), 0.5 mM IC87114

alone, (E) or in combination with 0.1 mM compound 15e (F). After different times, cells were lysed and processed for immunoblot analysis as described in

Materials and Methods. One representative experiment of two to three performed is reported.

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findings suggest that PI3Kg may trigger activation of class IAPI3Ks via Ras activation (Fig. 8). In light of the recent evidencethat PI3Kg lies downstream of Ras in neutrophils stimulated withfMLF (33) (Fig. 8), our findings point to the existence of a com-plex, positive-feedback circuit of Ras/PI3Kg/Ras activation. Alsothis mechanism of amplification of PI3K activities is likely in-dependent of SFKs. In fact, whereas early (1 min) phosphorylationof ERKs requires PI3Kg, a defect of ERKs phosphorylation isdetectable only at later time points in hck2/2fgr2/2lyn2/2 neu-trophils (Fig. 2) (i.e., when the early burst of ROS generation isceased) (Fig. 3). We conclude that SFKs play a nonredundant rolein activation of the early phase of neutrophil ROS generation inresponse to fMLF and play a minor role in the activation of classIA PI3Ks in this phase of the response (Fig. 8).The late, adhesion-dependent response to two independent

stimuli [i.e., fMLF (Fig. 4) and TNF (Fig. 5)] and upon stimulus-independent interaction with plastic, a b2 integrin–mediatedphenomenon (34, 39) (Fig. 5), is independent of PI3Kg expres-sion. However, the broad-specificity inhibitor wortmannin (Sup-plemental Figs. 1, 2) and more selective PI3Ka or PI3Kd (Fig. 6)inhibitory compounds totally or markedly suppressed adhesion-dependent ROS generation by human neutrophils, respectively.These inhibitors also hampered both stimulus-independent neu-

trophil spreading on plastic and TNF-induced human and murineneutrophil spreading on fibrinogen (Fig. 5, Supplemental Fig. 4,and data not shown). These findings show that class IA PI3Ks playan indispensable, albeit overlapping, role in adhesion-dependentneutrophil activation by adhesive surfaces. This is in concert withprevious studies that implicated class IA, but not class IB, PI3Ksin integrin-dependent ROS generation induced by neutrophil bindingof Aspergillus fumigatus hyphae (26). In this last study, PI3Kb wasshown to play a greater role than PI3Ka in the ROS and spreadingresponse to Aspergillus hyphae, whereas the TNF-induced response(Fig. 6) was less sensitive to PI3Kb rather than PI3Ka or PI3Kdinhibitors. One possible reason for this discrepancy may be that theinteraction of neutrophil with Aspergillus hyphae involves othersurface receptors in addition to b2 integrins (see Ref. 26 andreference contained therein). Notably, although deficiency ofPI3Kg did not result in a lack of late adhesion-dependentresponses (Figs. 4, 5), we found that AKT phosphorylation inresponse to fMLF (Fig. 2), but not TNF (Supplemental Fig. 3),was dependent on PI3Kg expression. This finding suggests thatat these late time points, there is not a simple relationship amongPI3K activity, PIP3 formation, and AKT phosphorylation. It istempting to speculate that chemoattractant signaling can, indepen-dently of the net effect on PIP3 formation, impinge on pathways

FIGURE 8. Model for signal transduction mechanisms regulating early, adhesion-independent, and late, adhesion-dependent neutrophil ROS generation.

Trimeric G proteins coupled to fMLF receptor(s) (fMLF-R) dissociate in a b/g heterodimer and a Gai subunit upon ligand–receptor interaction. The b/g

heterodimer activates PI3Kg that promotes formation of PIP3. The evidence that Ras activation and phosphorylation of the Ras downstream target ERK is

virtually absent in PI3Kg-deficient murine neutrophils (Figs. 2, 3) suggests that activation of GEFs for Ras by PIP3 may result in activation of class IA

PI3Ks. However, this scenario is complicated by the recent demonstration that G protein–coupled receptors impinge on PI3Kg via a PLCb/diacylglycerol/

RasGRP4 pathway (blue arrows) that plays a key role in PI3K-dependent neutrophil responses (33). Taken together, these findings point to the existence of

a positive-feedback circuit of PI3K activation implicating Ras/PI3Kg/Ras. Gai, b-arrestin bound to the receptor–G protein complex or the G protein–

coupled receptor directly, impinges on SFKs (43, 44). For the sake of simplicity, only the first possibility is highlighted in the drawing. Although

mechanism of activation of SFKs by trimeric G protein–coupled receptors in neutrophils is still unknown, several reports pointed to a role of SFKs in signal

transduction by this class of receptors (14, 38, 45–49). Activation of PI3Kg and SFKs are both essential for the early activation of NADPH oxidase by

fMLF, and either deficiency or inhibition of these kinases results in a marked or total suppression of ROS formation in murine or human neutrophils,

respectively (Fig. 4, Supplemental Fig. 1). Because inhibitors of the class IA PI3K isoforms PI3Ka and PI3Kd inhibit the early phase of ROS generation

(Fig. 6), it is likely that a positive-feedback circuit triggered by PI3Kg/Ras results in activation of class IA PI3Ks. Albeit, using selective inhibitors, we

found that PI3Kb plays a minor role in the early response to fMLF, this PI3K isoform has been implicated in the prolonged, late phase of ROS generation

by neutrophils adhering to Aspergillus hyphae or immune complexes (26, 50). It is likely that class IA PI3Ks play a highly redundant role in regulation of

this response. SFKs play a minor role in activation of class IA PI3Ks (broken arrow) in the early response to fMLF. In fact, inhibition of SFKs by PP2 in

human, or deficiency of Hck, Fgr, and Lyn in murine, neutrophils only slightly inhibits, or has no effect on, phosphorylation of the PI3K downstream target

AKT, respectively (Fig. 1). SFK inhibition (Supplemental Fig. 1) or deficiency (Fig. 4) result in a total or marked reduction of ROS generation. One

important target of SFKs is the Vav family of GEFs for Rac. Inhibition of SFKs by PP2 in human, or deficiency of Hck, Fgr, and Lyn in murine, neutrophils

results in a total inhibition of Vav phosphorylation (Fig. 7). Following interaction with chemoattractants, TNF, or other cytokines, neutrophils generate ROS

in a fashion characterized by lateness and b2 integrin dependency (6). This response is totally independent of PI3Kg (Figs. 4, 5), and requires class IA

PI3Ks and SFKs (Figs. 5, 6). SFKs regulate class IA PI3Ks in the course of this response (Fig. 1, Supplemental Fig. 3) and are essential for phosphorylation

of Vav proteins (Fig. 7).

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regulating AKT dephosphorylation. Clearly, this issue deservesfurther investigation.In the course of the late, adhesion-dependent phase of the neu-

trophil response, SFKs regulate class IA PI3Ks. In fact, in responseto both fMLF and TNF, the late phase of phosphorylation of thePI3K downstream target AKT was totally suppressed by PP2 inhuman neutrophils and reduced in hck2/2fgr2/2lyn2/2 neutrophils(Fig. 1, Supplemental Fig. 3). Hence, whereas in the early responseto chemoattractants PI3Ks and SFKs play a largely distinct, non-reduntant role, in the late adhesion-dependent response, activationof SFKs is also implicated in activation of class IA PI3Ks.Independently of their role in regulating PI3K activities, SFKs

deliver signals that are indispensable for NADPH oxidase acti-vation both in the early and late phase of ROS generation. Acti-vation of the small GTP-binding protein Rac is essential foractivation of NADPH oxidase (1, 16–18). Whereas PIP3 formationby PI3Ks regulates activation of at least three different GEFs forRac (see Ref. 23 and references contained therein), Vav familyproteins seem to be the only target of SFKs. Recent studieshighlighted an essential requirement for cooperation of P-Rex1and Vav proteins in the early response to chemoattractants (23)but confirmed previous studies assigning to Vav an exclusive rolein the regulation of responses depending on prolonged adhesion toIgs and integrin ligands (8, 27, 40, 41). SFKs play a central role inVav protein phosphorylation in both the early and delayed re-sponse to both fMLF and TNF (Fig. 7). This effect may be eitherdirect or, at least for the late, adhesion-dependent response, occurdownstream of Syk (8) or Pyk2 (42). In the light of the strongevidence implicating Vav proteins in activation of neutrophil ROSgeneration (19, 23), this suggests that one major mechanism bywhich SFKs regulate ROS generation is Vav protein phosphory-lation. Interestingly, we found that Vav phosphorylation is notabsolutely dependent on PI3Ks (Fig. 7). In fact, TNF-inducedactivation of delayed neutrophil responses is accompanied byVav phosphorylation also in the presence of wortmannin or morespecific class IA PI3K inhibitors. The clear dissociation betweeninhibition of ROS generation and Vav phosphorylation in responseto TNF by PI3K inhibitors (Fig. 7, Supplemental Fig. 3) suggeststhat other PI3K/PIP3-dependent Rac GEFs are also implicated inthis response (see Refs. 14, 23 and references contained therein),and SFK-dependent phosphorylation of Vav occurring downstreamintegrin engagement may be independent of PI3K activities.Mechanisms regulating neutrophil activation are attracting in-

creasing attention due to the high incidence of autoimmune dis-eases and the need to identify new targets to control inflammation.We identified SFKs as a key component of signal transductionensuing both from G protein–coupled receptors for chemoattrac-tants and from integrin receptors in the presence of TNF. PI3Ksact in a coordinated, nonredundant fashion with SFKs in regu-lating both type of the responses. Notably, PI3Kg is implicatedonly in the chemoattractant-induced response, whereas class IAPI3Ks, especially the PI3Ka and PI3Kd isoforms, are essential forthe regulation of the adhesion-dependent response. This scenariohighlights a rationale to define possible strategies to control differentforms of inflammatory responses and inhibit sterile inflammationavoiding excessive depression of host defenses.

DisclosuresThe authors have no financial conflicts of interest.

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